Extracellular vesicle, preparation method thereof, and typing analysis method

Abstract

The purpose of the present invention is to provide an extracellular vesicle, a preparation method thereof, and a typing analysis method. By removing the surface N-sugar of the extracellular vesicles, the uptake rate of the extracellular vesicles by specific cells is increased. This technology uses co-culture of living cells and extracellular vesicles, fluorescent labeling, and flow cytometry to detect fluorescent cells and fluorescence intensity in cell suspensions in different cell culture dishes. The interaction between living cells and extracellular vesicles is used to perform typing analysis and verification of the extracellular vesicles. In addition, the invention provides a method for preparing extracellular vesicles that can be more efficiently taken up by HCT116.

Description

Technical Field

[0001] The present invention relates to the field of extracellular vesicles, and in particular to an extracellular vesicle and a preparation method and a typing analysis method thereof. Background Art

[0002] Extracellular vesicles (EVs) are nanometer-sized, bilayer, spherical proteoliposomes with a particle size of approximately 50-100 nm. Their outer surface is surrounded by a phospholipid bilayer and is rich in water-soluble proteins, mRNA, and microRNA.

[0003] In terms of drug delivery, traditional drug carriers are large in size and toxic, relatively complex to synthesize, and easily phagocytosed by macrophages, preventing them from effectively reaching tumor sites. Traditional drug carriers include cationic liposomes and cationic polymers. Their permanent positive surface charge allows them to bind drugs through electrostatic interactions. However, after intravenous injection, cationic liposomes bind to negatively charged serum proteins, resulting in poor targeting and a lack of long-term circulation in the body. Extracellular vesicles, on the other hand, are membrane-bound vesicles that carry a large number of protein and carbohydrate macromolecules on their surface. Furthermore, their small size and excellent biocompatibility make them promising drug delivery vehicles. Molecular features on the surface of extracellular vesicles may determine how they interact with their target cells. Therefore, when using extracellular vesicles as drug carriers, it is essential to analyze which sources of extracellular vesicles interact best with target cells, or which have the best ability to target specific cells.

[0004] Currently, extracellular vesicle profiling is often performed using direct analysis of extracellular vesicle components, such as immunoblotting, mass spectrometry, sequencing, and flow cytometry. Some technologies rely on molecular-level extracellular vesicle profiling, which is costly and time-consuming. Extracellular vesicle uptake by target cells may occur through multiple molecular interactions, and in biorecognition, mutual recognition of molecules often depends on their physiological conformation. Most existing technologies use lysis to obtain extracellular vesicles, which cannot detect the binding of extracellular vesicles to specific cells while ensuring the activity of molecules on the extracellular vesicle surface, potentially losing a lot of information. Summary of the Invention

[0005] In response to the shortcomings of the existing technology, the purpose of the present invention is to provide a method for extracellular vesicle typing and analysis. At the same time, by modifying extracellular vesicles - removing the surface N-sugar of extracellular vesicles, the uptake rate of extracellular vesicles relative to specific cells is increased. This technology uses co-culture of living cells and extracellular vesicles, and utilizes the interaction between living cells and extracellular vesicles to perform typing analysis and verification of extracellular vesicles.

[0006] To achieve the above objectives, the present invention provides a method for extracellular vesicle typing and analysis based on uptake rate, comprising the following steps:

[0007] Step 1: Label different extracellular vesicles with fluorescent markers, remove excess fluorescent markers by ultracentrifugation, and resuspend the extracellular vesicles in 100 μL PBS;

[0008] Step 2: Add equal amounts of the different extracellular vesicles labeled in step 1 to different cell culture dishes and incubate with specific cells at 37°C and 5% carbon dioxide for 1.5-3 hours;

[0009] Step 3: Remove the culture medium and unadsorbed extracellular vesicles, wash with PBS, add 500 μL of trypsin to digest the cells, centrifuge, remove the supernatant, and resuspend the cells with PBS;

[0010] Step 4: Use flow cytometry to detect fluorescent cells and fluorescence intensity in cell suspensions in different cell culture dishes.

[0011] As a further improvement of the present invention, the specific cells in step 2 are HCT116 cells.

[0012] The present invention also provides a method for increasing the uptake rate of extracellular vesicles, comprising the following steps:

[0013] Step 1: Centrifuge and separate the extracellular vesicles, collect the extracellular vesicles, resuspend the precipitate with PBS to obtain the extracellular vesicle resuspension, and perform protein quantitative analysis of the extracellular vesicles using a BCA protein quantification kit;

[0014] Step 2: Add 1000 units of PNGF enzyme to 1 μg of extracellular vesicles, mix thoroughly, and incubate at 37°C for 2 hours;

[0015] Step 3: ultracentrifuge the extracellular vesicles in step 2, remove the supernatant, collect the precipitate, and resuspend the precipitate with PBS to obtain a resuspension of extracellular vesicles with N-glycans removed;

[0016] Step 4: The extracellular vesicle resuspension obtained in step 3 is quantified using a BCA protein quantification kit.

[0017] The present invention provides an extracellular vesicle, wherein the extracellular vesicle is an extracellular vesicle whose surface N-sugar is modified or enzymatically hydrolyzed.

[0018] As a further improvement of the present invention, the extracellular vesicles are selected from HCT116 extracellular vesicles.

[0019] The present invention also provides an application of extracellular vesicles as drug carriers.

[0020] As a further improvement of the present invention, the exosomes are anchored or injected into the surface or interior of a carrier to form a shape-fixed, implantable complex. After the complex is implanted, the exosomes therein are released in a sustained, directionally or controllably manner; the complex includes various prostheses or stents.

[0021] As a further improvement of the present invention, extracellular vesicles are anchored or injected into the surface or interior of a carrier to form a dressing with a fixed shape. After the dressing adheres to the tissue, the extracellular vesicles therein can be slowly, directionally, and controllably released into the tissue.

[0022] As a further improvement of the present invention, the extracellular vesicle suspension is prepared as a cell culture additive for transporting substances to specific cells.

[0023] The beneficial effects of the present invention are: by modifying or enzymatically degrading the surface N-glycans of extracellular vesicles, a type of extracellular vesicle that can be rapidly taken up by specific cells is studied and prepared. When applied to tumor drug carriers, it can play a targeting role and can increase the rate of drug uptake, thereby increasing the drug concentration taken up by target tumor cells per unit time. By disclosing an experimental method that can determine which type of extracellular vesicles a specific cell has a stronger affinity for, it is possible to achieve typing analysis of extracellular vesicles. This can be used to verify and screen the extracellular vesicles with the highest binding to specific cells after different modifications to the outer surface of the cell vesicles. The present invention maximizes the activity of molecules on the surface of extracellular vesicles, can reflect the interaction between extracellular vesicles and specific cells, and can reflect the results of multiple molecular interactions. In addition, in the natural state of the molecules, it can also reflect the conformation-dependent interaction mode of different molecules. It provides more multi-dimensional information on the composition of extracellular vesicles than simply from the molecular spectrum. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 Flow cytometry graphs of the extracellular vesicles of the experimental group and the control group after co-culture with HCT116 cells in Example 2 of the present invention. DETAILED DESCRIPTION

[0025] The present invention will be further described in detail below with reference to the accompanying drawings.

[0026] In Example 1, the present invention provides a method for increasing the uptake rate of extracellular vesicles, comprising the following steps:

[0027] Step 1: Extract HCT116 extracellular vesicles by differential centrifugation: After adding the sample to the centrifuge tube, centrifuge at 500g for 15 minutes and take the supernatant. Transfer the supernatant to a new centrifuge tube and centrifuge at 2000g for 15 minutes. Transfer the supernatant to a new centrifuge tube and centrifuge at 10000g for 15 minutes; transfer the supernatant to an ultracentrifuge tube and centrifuge at 120000g for 2 hours. Remove the supernatant, collect the precipitate, and resuspend the precipitate with PBS to obtain the extracellular vesicle resuspension. The extracellular vesicle resuspension is quantified using the BCA protein quantification kit;

[0028] Step 2: Add 1000 units of PNGF enzyme to 1 μg of extracellular vesicles, mix thoroughly, and incubate at 37°C for 2 hours;

[0029] Step 3: Place the extracellular vesicles obtained in step 2 into an ultracentrifuge tube, centrifuge at 120,000 g for 2 hours, remove the supernatant, collect the precipitate, and resuspend the precipitate with PBS to obtain a resuspension of extracellular vesicles with N-glycans removed; Step 4: Quantify the extracellular vesicles in the resuspension obtained in step 3 using a BCA protein quantification kit.

[0030] In Example 2, the present invention provides an extracellular vesicle typing analysis method based on uptake rate, wherein the extracellular vesicles prepared according to Example 1 are used as an experimental group and HCT116 extracellular vesicles not treated with PNGF are used as a control group to measure the binding rate of the two extracellular vesicles to HCT116 tumor cells, comprising the following steps:

[0031] Step 1: Take 20 μg of extracellular vesicles from the experimental group and the control group, label the membrane of the extracellular vesicles of the two groups with PKH67 dye, add 3% BSA to terminate the reaction, remove excess PKH67 dye by ultracentrifugation, and resuspend the extracellular vesicles in 100 μL PBS;

[0032] Step 2: Add equal amounts of the different extracellular vesicles labeled in step 1 to HCT116 cell culture medium and incubate with HCT116 cells at 37°C and 5% carbon dioxide for 1.5 h or 3 h;

[0033] Step 3: After the culture is completed, remove the culture medium and unadsorbed extracellular vesicles, wash with PBS, add 500 μL of trypsin to digest the cells, centrifuge, remove the supernatant, and resuspend the cells with PBS;

[0034] Step 4: Flow cytometry was used to detect the number of fluorescent cells and the fluorescence intensity in the cell suspensions in different HCT116 cell culture dishes after 1.5 hours and 3 hours, and a blank control was used without fluorescently labeled extracellular vesicles.

[0035] like Figure 1 As shown in the figure, after 1.5 hours of co-incubation of extracellular vesicles with HCT116 cells, HCT116 cells took up more HCT116 extracellular vesicles with N-glycans removed, while after 3 hours of incubation, the difference in the amount of HCT116 extracellular vesicles taken up by HCT116 cells was smaller than that of HCT116 extracellular vesicles with N-glycans removed. This experiment shows that HCT116 cells have a higher efficiency in taking up HCT116 extracellular vesicles with surface N-glycans removed than those without.

[0036] The HCT116 extracellular vesicles with N-glycan removed prepared above are applied to a drug carrier system. By increasing the uptake rate of the extracellular vesicles, the anti-tumor drug can be efficiently loaded into the extracellular vesicles when applied to the drug carrier system.

[0037] The specific steps are as follows: the prepared N-glycan-depleted HCT116 extracellular vesicles are dispersed in PBS buffer, and dodecyl maltoside is added to the solution at a mass ratio of 10:10:1000. The mixture is shaken at 37°C for 10 hours. An anti-tumor drug is then added to the N-glycan-depleted HCT116 extracellular vesicle solution, and the mixture is incubated at 37°C for 10 hours. The anti-tumor drug can gradually diffuse from a high concentration gradient to a low concentration gradient, entering the N-glycan-depleted HCT116 extracellular vesicles. To prevent drug leakage, calcium chloride ions are added to the solution to a concentration of 1 mg / ml. The solution is incubated at 37°C for another 10 hours, followed by ultracentrifugation. The supernatant is discarded, and the pellet is resuspended in PBS buffer. Repeat ultracentrifugation at 200,000 g for 5 h, discard the supernatant, and resuspend the precipitate in PBS buffer to obtain extracellular vesicles carrying anti-tumor drugs.

[0038] When used as a drug carrier, the extracellular vesicles are anchored or injected into the surface or interior of the carrier to form a complex with a fixed shape that can be implanted. After the complex is implanted, the extracellular vesicles therein are released slowly, directionally or controllably; the complex includes various prostheses or scaffolds.

[0039] Alternatively, the extracellular vesicles can be anchored or injected into the surface or interior of a carrier to form a dressing with a fixed shape. After the dressing adheres to the tissue, the extracellular vesicles therein can be released into the tissue in a sustained, directed, and controllable manner.

[0040] Alternatively, the extracellular vesicle suspension can be prepared as a cell culture additive for transporting substances to specific cells. The above are only preferred embodiments of the present invention. The scope of protection of the present invention is not limited to the above embodiments. All technical solutions under the concept of the present invention fall within the scope of protection of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications without departing from the principle of the present invention should also be considered as the scope of protection of the present invention.

Claims

1. A method for increasing the rate of uptake of HCT116 extracellular vesicles by HCT116 cells, characterized in that: The method comprises the following steps: step 1, separating extracellular vesicles by centrifugation, collecting the extracellular vesicles, resuspending the precipitate with PBS, and performing protein quantitative analysis of the extracellular vesicles using a BCA protein quantitative kit; Step 2: Add 1000 units of PNGF enzyme to 1 μg of extracellular vesicles, mix thoroughly, and incubate at 37°C for 2 hours. Step 3: ultracentrifuge the extracellular vesicles in step 2, remove the supernatant, collect the precipitate, and resuspend the precipitate with PBS to obtain a resuspension of extracellular vesicles with N-glycans removed; Step 4: The extracellular vesicle resuspension obtained in step 3 is quantified using a BCA protein quantification kit.

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